Elevated NO production has been detected in patients suffering from various arthropathies; however, its role and regulation during gouty arthritis remain largely unexplored. Monosodium urate (MSU) crystals, the causative agent of gout, have been shown to induce NO generation in vivo and inducible NO synthase (iNOS) expression in human monocytes. The present study was designed to evaluate the ability of MSU crystals to modulate macrophage (Mφ) iNOS expression and NO synthesis and to investigate the molecular mechanisms underlying these cellular responses. We found that MSU crystals did not induce NO production in murine J774 Mφ. However, a synergistic effect on the level of iNOS expression and NO generation was observed in cells exposed to MSU crystals in combination with IFN-γ. Characterization of the second messengers involved revealed the requirement of IFN-γ-mediated Janus kinase 2/STAT1α activation even though MSU crystals did not modulate this signaling cascade by themselves. MSU crystals exerted their up-regulating effect by increasing extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and NF-κB nuclear translocation in response to IFN-γ. The use of specific inhibitors against either NF-κB or the ERK1/2 pathway significantly reduced MSU+ IFN-γ-inducible NF-κB activity, iNOS expression, and NO production. Altogether, these data indicate that MSU crystals exert a potent synergistic effect on the IFN-γ-inducible Mφ NO generation via ERK1/2-and NF-κB-dependent pathways. Understanding the molecular mechanisms through which MSU crystals amplify Mφ responses to proinflammatory cytokines such as IFN-γ will contribute to better define their role in NO regulation during gout, in particular, and inflammation, in general.